Project Summary/Abstract: Neural tube closure is a necessary stage of vertebrate embryonic development during which the central nervous system is formed. Failures of neural tube closure, termed neural tube closure defects, are common and serious birth defects that can be either debilitating or fatal. Despite the importance of neural tube closure to vertebrate development, little is known about the morphogenetic cell behaviors underlying this process. One important process in neural tube closure is apical constriction, where the epithelial cells shrink their apical surfaces through actomyosin contractions, facilitating tissue bending and proper closure of the neural tube. Failure of apical constriction results in lethal neural tube defects such as anencephaly. Several genes regulating apical constriction in the neural plate have been identified, but it is unknown how various modes of actomyosin contractions contribute to apical constriction and how apical constriction genes contribute to those modes of actomyosin contractions. This proposal contains experiments that will quantitatively describe actomyosin contractions and apical constriction across the neural plate during neural tube closure. This will be achieved thorough live-imaging of cell shape and cell behavior via cell junctions, and actomyosin dynamics via actin and myosin. Data on cell behavior and actomyosin will then be used to map apical constriction behaviors spatially and temporally to the neural plate in order to understand how various modes of actomyosin contraction contribute to neural tube closure. In addition, it is proposed to examine how the neural tube closure genes Shroom3 (necessary for apical constriction) and N-cadherin (necessary for adhesion) contribute to actomyosin contractions during neural tube closure. Gene function will be assessed by using CRISPR/Cas9 mutatgenesis to generate both mosaic and tissue-wide mutants for both Shroom3 and N-cadherin. CRISPR/Cas9 will also be used to generate fluorescent-labeled endogenous N-cadherin in order to mark cell junctions and examine N-cadherin dynamics in live cells. Overall, this proposal aims to both quantitatively analyze a little-studied cell behavior which is necessary to neural tube closure and implicated in serious birth defects. In addition, molecular tools and strategies will be developed to aid study of neural tube closure in proposed experiments and into the future.